Plant Physiol. (1970) 46, 806-811

Control of Ribonuclease and Acid Phosphatase by Auxm andAbscisic Acid during Senescence of Rhoeo Leaf Sectionsl

PIETRO DE LEO2 AND JOSEPH A. SACHERDepartment of Botany, California State College, Los Angeles, California 90032

ABSTRACT

We report the effects of abscisic acid and auxin (a-naph-thalene acetic acid) on regulation of enzyme synthesisduring senescence of leaf sections of Rhoeo discolor Hance.Abscisic acid always accelerates the onset of and enhancesthe magnitude of the increase in activity of acid phospha-tase; this is followed by an acceleration of the onset of arapid increase in free space.RNase activity increases 2- to 5-fold after cutting of leaf

sections. Abscisic acid increases RNase activity and inhibitsthe rate of incorporation of uridine and leucine in leaf sec-tions removed from plants grown under stress but notfavorable conditions. Auxin inhibits the increase in RNaseand acid phosphatase and suppresses the effects of abscisicacid. The increase in activity of RNase and acid phosphataseis inhibited by inhibitors ofRNA and protein synthesis. Thisand other evidence suggests that the increases in hydrolaseactivity could result from new enzyme synthesis. The pos-sible significance of the results in respect of hormonal regu-lation of enzyme activity and senescence is discussed.

Auxin prevents senescence of Rhoeo leaf sections maintained onendogenous substrates in darkness (12), prevents degradation ofprotein, and enhances incorporation of uridine and leucine in allsubcellular fractions (14). These effects could be attributable toeither an effect of auxin on preventing degradation or stimulatingsynthesis of RNA and protein. It has been demonstrated from anumber of investigations that hydrolase activity increases duringaging of tissues of determinate organs. For example, RNase ac-tivity increases with age of apple leaves (9), or as a result of sub-jecting leaves to water stress (5), or vacuum infiltration of water(2). Activity of two phosphatases increases in excised Avena leaves(20). Also, a number of recent reports indicate that treatment ofexcised tissues with kinetin or abscisic acid (18, 21) and auxin(16, 19) affect the activity of certain hydrolytic enzymes.The present study was directed toward elucidation of the possi-

ble role of hydrolases in respect of hormonal regulation of senes-cence. We report on the senescent changes which occur duringaging of Rhoeo leaf sections involving increased activity of thehydrolytic enzymes RNase and acid phosphatase, and the effectsof auxin or abscisic acid on regulation of the activity of thesehydrolases.

1 This investigation was supported by National Science FoundationGrant GB-8316 to J. A. Sacher.2On leave from Laboratorio de Radiobiochimica ed Ecofisiologia

Vegetale, C.N.R., Roma, Italy, with a fellowship supported by theNorth Atlantic Treaty Organization. Present address: Department ofBotany, University of Bari, Bari, Italy.

MATERLALS AND METHODS

Fully expanded leaves of Rhoeo discolor Hance. were surfacesterilized in 0.5% sodium hypochlorite for 15 min, and then sec-tions (1.5 x 10 mm) were cut from the basal midrib region. Thesections were washed in running water for 15 min to remove ma-terials released from cut surfaces. The sections were blotted, andduplicate samples (500 mg of fresh weight) were weighed andaged for 4 to 72 hr in the dark at 22 C in Petri dishes on filterpaper wetted with a solution of 25 ,g/ml streptomycin sulfate(to aid in preventing microbial contamination), with and withoutABA or auxin (a-naphthaleneacetic acid3). For some experiments,inhibitors of synthesis of RNA (6-methylpurine, chromomycinA3 or 5-fluorouracil) or protein (cycloheximide) were added to thesolutions. Chromomycin As strongly inhibits DNA dependentRNA polymerase activity (8) and inhibits incorporation of RNAprecursors in Rhoeo leaf sections (14).

Radioassay. For measurement of the rate of synthesis of RNAand protein, samples of tissue sections were incubated in solutionsdual labeled with uridine-5-T and L-leucine-U-'4C (Schwarz Bio-research), containing 25 ,ug/ml of streptomycin sulfate and werebuffered to pH 6.0 with 0.025 M phosphate buffer. The sectionswere washed, homogenized, and extracted for assay of the ratioof dpm incorporated per dpm total uptake as described (15).Enzyme solutions were prepared by homogenizing the tissue

samples in a Potter-Elvehjem tissue grinder at 2 to 4 C in 2 ml ofgrinding medium containing 0.1 M acetate buffer, pH 5.0, and7% Carbowax 4000 (Union Carbide Corp.), to prevent inactiva-tion of enzymes (22).

Ribonuclease. For assay of RNase the crude homogenates werecentrifuged at 10,000g at 2 C, and the supernatant fraction wasused as a source of enzyme. Enzyme reaction mixtures of 0.2-mlvolume containing enzyme (25 mg of fresh weight equivalent), 7%Carbowax 4000, 0.1 M acetate buffer, pH 5.0, and 400 ,gg of highlypolymerized yeast RNA (Calbiochem) labeled with 0.2 ,c of"4C-labeled yeast RNA (International Chemical and NuclearCorporation) were incubated at 40 C. At zero time and at the endof assays, a 0.1-ml aliquot was removed, mixed with 0.9 ml ofabsolute ethanol, chilled to 5 C for 2 hr, centrifuged, and theradioactivity was determined in aliquots of the ethanol solublefraction with a liquid scintillation spectrometer. Corrections weremade for quenching with an internal standardization method.Assay was based on the amount of ethanol soluble 14C-nucleo-tides formed, after correction for ethanol soluble radioactivitypresent at zero time. The accuracy of the foregoing method waschecked with an alternate method for measuring the amount ofRNA hydrolyzed. Toward this end, RNase activity was measuredbased on direct assay, at 260 nm, of acid soluble nucleotidesformed in presence of the enzyme, by the method of MacFayden(11). The percentage of hydrolysis of RNA was determined withthe MacFayden method and the "C-labeled RNA method did notvary significantly.

3Abbreviations: NAA: a-naphthalene acetic acid; 5-FU: 5-fluoro-uracil.

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HORMONAL CONTROL OF RNASE AND ACID PHOSPHATASE

Acid Phosphatase. For acid phosphatase the crude ho-mogenates were centrifuged at 30,000g. The supernatant fractionwas used as a source of soluble enzyme. The precipitate fractionwas washed once with a grinding medium, centrifuged, and thesupernatant fraction was discarded. The precipitate was extractedwith 2 ml of grinding medium containing 1% Triton X-100, andcentrifugal supernatant fraction was used as an enzyme source.Enzyme reaction mixtures of 5-ml volume containing 0.1 M, pH5.0 acetate buffer, 7% Carbowax 4000, 0.8 mg p-nitrophenylphos-phate, and enzyme solution (100 mg of fresh weight equivalent)were incubated at 40 C. At zero time and at intervals, 1 ml wasremoved and mixed with 3 ml of 0.168 N NaOH, and the absorb-ance at 400 nm was determined after 10 min. All enzyme assayswere made during the initial linear phase.

Free Space. Free space was determined based on measurementof the dilution of radioactivity in a 0.15 M '4C-mannitol solutionin which tissue sections were bathed, owing to diffusive movementof radioactivity into the tissue as described (13, 14).

RESULTS

Effect of ABA on Senescence of Rhoeo Leaf Sections. ABAgreatly accelerates senescence of Rhoeo leaf sections, as it does ofother leaf tissues (6, 17). Incubation of sections in 0.001 mm ABAcaused loss of membrane integrity and was manifested by leakageof solutes and intense browning of tissue 2 days earlier than inwater controls. Addition of auxin inhibited the effects of ABA.The quantitative effect ofABA on membrane permeability and

senescence can be illustrated by measurements of changes in thepercentage of the tissue volume that is free space to 0.15 M 14C-labeled mannitol (13, 14). When leaf sections were incubated in0.05 mM ABA, the free space increased rapidly to about 90% be-tween 24 and 48 hr, while in the water controls there was nochange in free space for 48 hr, after which it increased slowly (Fig.1). Auxin completely prevented the increase in free space (14) andinhibited the effect of ABA.

Following are the results of experiments used to ascertainwhether the acceleration of senescence is owed to ABA directlyinhibiting the synthesis ofRNA or protein.

Effects of ABA on Synthesis of Total RNA and Protein underDifferent Growth Conditions. Our first studies of the effect ofABA on synthesis of total RNA and protein were done with leafsections removed from plants grown at the Earhart Laboratory,California Institute of Technology, under conditions of tempera-

F

-x

,-

~T/

/.

rw /_

50 // --.-

40'~ ~_ 0 NAA_Qo~

0 24HOURS

48

FIG. 1. Comparison of the effects of ABA or NAA on changes infree space during aging of Rhoeo leaf sections for 72 hr. Procedure forassay of free space given under "Materials and Methods."

Table I. Effect of ABA and Auxin on Incorporation of Leucineand Uridine in Sections from Vigorously Growing LeavesDuplicate samples of Rhoeo (500 mg fresh wt) leaf sections were

aged 20 hr in Petri dishes at 22 C in 2.5 ml of water or solutions ofABA or NAA and then for another 6 hr in solutions dual-labeledwith leucine and uridine. All solutions contained 25 ,Ag/ml strep-tomycin and 0.025 M phosphate buffer, pH 6.0. ABA was 0.005mm and NAA 10,ug/ml. All differences in the data are significantat the 0.1% level or better.

Ratio of dpm Incorporated/dpm Total UptakeTreatment

Leucine Uridine

H20 0.447 0.430ABA 0.470 (100)1 0.370 (86)NAA 0.595 (133) 0.572 (133)

1 Numbers in parentheses show the ratio in percentage of thewater control.

Table II. Effect ofABA or Auxin on Incorporation of Leucine andUridine in Sections from Leaves Grown under Suboptimal

ConditionsProcedures as in Table I. ABA was 0.005 mm and NAA 10 ;g/ml.

All differences in the data are significant at the 0.1% level orbetter.

Ratio of dpm Incorporated/dpm Total UptakeTreatment

Leucine Uridine

H20 0.611 0.463ABA 0.264 (43)1 0.150 (32)ABA + NAA 0.495 (81) 0.445 (100)NAA 0.530 (87) 0.515 (111)

1 Numbers in parentheses show the ratio in percentage of thewater control.

ture, light, water, and nutrition favorable for vigorous growth(as was the previous work on Rhoeo leaves [12, 14]). Under thesefavorable conditions, ABA had no effect on the rate of incorpora-tion of leucine and inhibited incorporation of uridine 14% (TableI), while auxin enhanced synthesis of RNA and protein as re-ported previously (14).When controlled environment facilities at Earhart Laboratory

were closed down, Rhoeo plants were transferred in winter to atemporary greenhouse in which only suboptimal conditions oftemperature, light intensity, and light duration could be main-tained, which resulted in some retardation of the rate of growth.Several experiments showed that leaf sections removed fromplants grown under these suboptimal conditions were very sensi-tive to ABA. As illustrated in Table II, 0.005 mm ABA inhibitedthe rate of uridine incorporation 68% and leucine 57%. Concen-tration activity studies showed that the inhibition of incorpora-tion of uridine was 61, 75 and 84%, and of leucine 32, 52 and65% for 0.001 mm, 0.005 mm, and 0.01 mm ABA, respectively.For both labeled precursors, 0.01 mm ABA gave maximal in-hibition; 0.0001 mm had no effect. In leaf sections removed fromplants grown under suboptimal conditions, auxin caused only asmall stimulation of incorporation of uridine (11%) and in-hibited the rate of leucine incorporation; yet under these unfavor-able environmental conditions, auxin completely prevented theinhibition of incorporation of uridine by ABA and greatly di-minished the inhibitory effect ofABA on incorporation of leucine(Table II).

90

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DE LEO AND SACHER

The effect of ABA on incorporation of uridine and leucine wasassayed subsequently in May and June, with leaf sections fromvigorously growing plants. ABA had no effect on incorporationof uridine and leucine. ABA, however, had the same effect onaccelerating the increase in free space in leaf sections removed fromvigorously growing as from slowly growing plants. These observa-tions indicate that the inhibition of synthesis of total RNA andprotein by ABA may contribute, but is not essential, to the ac-celeration of senescence by ABA.

It could be demonstrated that when plants were subjected tolow nutrients and water, ABA inhibited incorporation of RNAand protein precursors, even though light and temperature con-ditions were very favorable. Thus, it appeared that the efficacy ofABA on inhibition of the rate of incorporation of both uridineand leucine may be enhanced by stress conditions of differentorigins.

Effect ofABA or Auxin on RNase. During aging of leaf sectionsfrom slowly growing plants for 24 hr, there occurred a 3- to 5-foldincrease in RNase activity (Table III), and this increase was en-hanced 48% by addition of 0.005 mm ABA. Auxin suppressed theincrease in RNase 38% as compared to the water controls andcompletely inhibited the effect of ABA on RNase. The amount ofRNase activity at zero time varied for different batches of tissue,

Table III. Effect of ABA or Auxini ont RNase Activity ill Sectionsfrom Leaves Growni under Suboptimal Conditions

Duplicate 500-mg samples of leaf sections aged 24 hr as de-scribed in "Materials and Methods" were washed, homogenized,centrifuged at 2 C, and the l0,OOOg supernatant fraction was usedas the enzyme source. Enzyme reaction mixture of 0.2-ml volumecontained enzyme, 400 ,ug of highly polymerized yeast RNA,0.2 ,uc 14C-labeled, highly polymerized yeast RNA, and 7% Carbo-wax 4000 buffered to pH 5.0 with 0.1 M acetate buffer. Assay pro-

cedure described in "Materials and Methods." ABA was 0.005mM and NAA, 10,ug/ml. All differences in the data are significantat the 0.1% level or better.

RNA Hydrolyzed

Treatment

Zero time 24-hr aged

% per 15 min per 25 mg fresh wt

H20 2.75 14.84NAA 10.25 (-38%)'ABA 20.62 (+48%)ABA + NAA 11.20 (-30%,)

'Numbers in parentheses show the effect of the treatments on

the development of RNase activity as compared with the water

controls.

20r

a

w

N

-J0

cr

a

4tz

15

l0

5

--- - 00 ~ ~ ~

0I,- AM0

~ o/

0c

0 3 6 10 24HOURS

FIG. 2 Time course study of the effect of ABA on RNase activity inexcised sections of Rhoeo leaves. Sections were aged and RNase as-sayed as described in Table III.

Table IV. Effect of Cycloheximide, 5-FU and 6-Methylpurine onthe Development of RNase in Tissue Sections

Cycloheximide was 10 ,g/ml, 6-methylpurine was 0.5 mm and5-FU, 2.0 mm. Enzyme assays as in Table III.

RNA hydrolyzedTreatment

Zero time 4-Hr aged

% per 15 min per 25 mg fresh wi

H20 7.3 16.5Cycloheximide 7.755-FU 13.106-Methylpurine 6.8

ranging from 2.5 to 8.3 %, with a mean for nine experiments of5.2% hydrolysis in the standard enzyme reaction mixture.Time course studies (Fig. 2) indicate that most of the increase

in RNase occurs during the first 6 hr after cutting of tissue sec-tions. Thus, it appears that the increase is triggered by wounding,just as other forms of stress induce an increase in RNase activity(2, 5). ABA has no effect on RNase during the first 3 hr and in-creased RNase activity only 15% between 3 and 6 hr. Other experi-ments showed that 10 ,ug/ml of NAA does not suppress the in-crease in RNase that occurs during the first 3 or 4 hr.

In leaf sections obtained from plants grown under stress condi-tions, the enhancement of RNase activity by ABA is accom-panied by a large inhibition of synthesis of RNA and protein(compare tables II and III). Although the cause and effect rela-tions between these two effects of ABA are not clear, it seemsthat under these conditions the enhancement of RNase activity byABA could hasten senescence. In three experiments done withleaf sections removed from vigorously growing plants, however,ABA did not increase RNase activity over that of the water-agedcontrols during 24 hr. As pointed out above, ABA acceleratedsenescence of leaf sections removed from plants grown underfavorable or suboptimal conditions. Thus, it seems unlikely thatthe only role of ABA in accelerating senescence is owed to aneffect on RNase.We have found no effect of auxin or ABA on RNase activity

when present at various concentrations during grinding pro-cedures and enzyme assay. Thus, it appears that these hormonesare not acting directly as allosteric effectors of RNase. Also, it isunlikely that auxin suppression of RNase activity is owed to thepresence of an inhibitor, since dilution of the enzyme solution,with substrate concentration kept constant, resulted in no increasein RNase activity per unit of enzyme. There are no factors in thesupernatant fraction that affect activity of the precipitate enzyme,as the activity of mixtures of enzyme from both fractions isadditive. The rapid increase in RNase in freshly cut tissue duringthe initial 4 hr is completely suppressed by cycloheximide or 6-methylpurine, whereas 5-FU inhibited RNase activity only 37%(Table IV).

Effect of ABA or Auxin on Acid Phosphatase Activity. Whenleaf sections are aged overnight in presence or absence of ABA,there is normally no change in the amount of acid phosphatase ina supernatant fraction of tissue homogenates (Table V). Infre-quently (in 2 of 11 experiments), we have observed an increase(about 33%) in the supernatant acid phosphatase. Precipitatefractions were washed once in grinding medium, prior to enzymeassay, for removal of residual soluble enzyme and then extractedwith Triton X-100. In Triton X-100 extracts, acid phosphatasewas increased 135% by ABA as compared with the zero timecontrols (Table V), but during this period of aging (20 hr) therewas no increase in enzyme activity in the Triton extracts of water-aged tissue. A concentration activity study showed that ABA en-hanced activity of the Triton-extractable enzyme 0, 170, 282, and

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HORMONAL CONTROL OF RNAsE AND ACID PHOSPHATASE

Table V. Effect of ABA on Acid Phosphatase Activity of RhoeoLeaf Sections

Duplicate 500-mg fresh wt samples of leaf sections aged 20 hras described in "Materials and Methods," were washed, homoge-nized, centrifuged at 2 C, and the supernatant fraction used as anenzyme source. The ppt fraction was washed once in 0.1 M acetatebuffer, pH 5, and then was extracted with 0.1 M acetate buffercontaining 1% Triton X-100, centrifuged, and the supernatantfraction was assayed for acid phosphatase as in "Materials andMethods" section. The substrate was p-nitrophenylphosphate.ABA was 0.005 mM.

Treatment Supernatant of Triton Extract of ppt30,0OOg

A per 10 min per 20 mg fresh wt

Zero time 0.920 0.07420 hr H20 0.900 0.054 (100)220 hr ABA 0.960 0.174 (235)

1 For Tables V through IX, A X 0.207 = ,moles p-nitrophenyl-phosphate hydrolyzed per 10 min per 20 mg fresh wt.

2 Figures in parentheses show enzyme activity in percent of thezero time control.

Table VI. Distribution of Acid Phosphatase anid Effect of ABA onAcid Phosphatase in the Precipitate Fractiont in Respect of

Tritonz X-100 ExtractabilityThe 30,000g precipitate fraction of a tissue homogenate was

washed once in grinding medium. The remaining precipitate wasextracted twice with grinding medium containing 1% Triton X-100, and each of these supernatant fractions was assayed for acidphosphatase as described in "Materials and Methods." Thefinal precipitate fraction was incubated in the enzyme assay me-dium to determine enzyme activity remaining. ABA was 0.05 mm.All differences are significant at better than the 0.1% level.

Trssueatmentiof 1st Triton Extract 2nd Triton Extract Final ppt FractionTissue Sections

A per 10 min per 20 mg fresh wt

Zero time 0.094 0.030 0.78048 hr H20 0.136 (144)' 0.050 (167) 0.630 (81)48 hr ABA 0.420 (446) 0.130 (434) 0.820 (100)

1 Figures in parentheses indicate enzyme activity in percent ofthe zero time controls.

320% in 24 hr, at concentrations of 0.0005 mM, 0.005 mM, 0.05mM,and 0.5 mm, respectively. There was, however, no difference in theeffect of 0.001 and 0.05 mM ABA in accelerating the onset andrate of the increase in free space, which is an irreversible senescentchange.An analysis of the precipitate from tissue aged for 48 hr (Table

VI) shows that the fraction of acid phosphatase that is extractedwith Triton increases in water-aged tissue, and this increase is en-hanced 3-fold by ABA. In contrast, there is no increase in theacid phosphatase remaining with the precipitate after two suc-cessive extractions with Triton in presence or absence of ABA.A further analysis of the precipitate fractions from tissue ho-

mogenates by differential centrifugation shows that 75% of theTriton-extractable acid phosphatase is obtained from a 500g pre-cipitate (Table VII), which would contain cell wall debris, nuclei,chloroplasts, and some membrane material. The remaining 25%is recovered in the precipitate resulting from centrifuging thesupernatant fraction at 12,000g. Most (over 60%) of the acidphosphatase that may be extracted from the washed precipitatewith Triton X-100 is also extractable with 0.2 M CaCl

-

or NaCl.

There is no effect of tonicity (from 0.05 to 0.4 M) of the grindingmedium on the distribution of acid phosphatase in the soluble andprecipitate fractions.A time course study shows that ABA caused the activity of the

Triton-extractable acid phosphatase to increase 24 hr earlier thanin water-aged tissue (Fig. 3). Auxin, which prevents senescence ofRhoeo leaf sections (12, 14), completely suppresses the increase inacid phosphatase, and also suppresses the ABA-induced increasealmost completely during the first 24 hr and about 50% duringthe next 24 hr (Fig. 3). The increase in acid phosphatase in water-aged tissue and the effect of ABA in speeding the onset and mag-nitude of this increase are consistent, irrespective of whether theleaf sections are removed from plants grown under favorable orsuboptimal conditions.

Effect of Inhibitors on Acid Phosphatase Activity. The increasein acid phosphatase that occurs between 24 and 48 hr (Fig. 3) inwater-aged tissue is inhibited about 75% by cycloheximide or 6-methylpurine (Table VIII). The ABA-induced increase in acidphosphatase activity in the precipitate fraction was completelyinhibited by cycloheximide in a 24-hr period; in fact, cyclohexi-mide decreased the level of acid phosphatase activity to about50% of that in the zero time controls (Table IX). ChromomycinA3 inhibited the ABA-induced increase in enzyme in the precipi-tate fraction 60% and 6-methylpurine inhibited it 100%. Thesupernatant enzyme appears to be quite stable and have a lowrate of turnover, as cycloheximide had no significant effect on itsactivity during the 24-hr period. In other experiments the effect ofinhibitors on the ABA-induced increase in acid phosphatase ac-tivity was determined during a shorter exposure. A 76% increase

Table VII. Localization of the Triton-extractable Acid Phosphataseby Differential Centrifugation

Tissue sections were aged for 24 hr in 0.5 mm ABA before ex-traction of enzyme.

Centrifugal Fraction A per 10 min per 20 mg fr wt

500g SN' 0.800Triton-extract of 500g ppt 0.31412,000g SN 0.620Triton-extract of 500-12,000g ppt 0.09030,000g SN 0.630Triton-extract of 12,000-30,000g ppt 0.000

1 SN: Supernatant fraction.

E.Q2E00

cio

0 7 24 48 72HOURS

FIG. 3. Time course of the development of acid phosphatase inpresence and absence of ABA or NAA. Acid phosphatase is extractedfrom a 30,000g ppt with 1% Triton X-100. Enzyme extraction andassay procedures described in Table V and in "Materials and Methods."

Plant Physiol. Vol. 46, 1970 809

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8Plant Physiol. Vol. 46, 1970

Table VIII. Effect of Cycloheximide and 6-Methylpurine on De-velopment of Acid Phosphatase in the Precipitate Fraction of

Water-aged TissueTissue sections aged for 24 hr in a 25 ,ug/ml solution of strepto-

mycin were washed and then transferred to fresh solutions inpresence and absence of cycloheximide (10,g/ml) and 6-methyl-purine (0.5 mM). The precipitate fraction was extracted andassayed as in experiments shown in Table V. All differences in thedata are significant at the 0.1% level or better.

Treatment A per 10 min per 20 mg fr wt

Zero time 0.12224 hr H20 0.110 (90)148 hr H20 0.436 (358)24 hr H20 + 24 hr cycloheximide 0.204 (167)24 hr H20 + 24 hr 6-methylpurine 0.184 (151)

1 Figures in parentheses express enzyme activity in percent ofzero time.

Table IX. Effect of Inhibitors on Acid Phosphatase Activity in theSupernatant and Precipitate Fractions

Tissue samples were aged and prepared for enzyme assay asdescribed in Table V, except inhibitors added as indicated. ABAwas 0.005 mM, 6-methylpurine, 0.5 mm, and cycloheximide, 10,ug/ml, chromomycin A3, 100,ug/ml. All differences in the dataare significant at the 1% level or better.

Treatment ~ ~ Spernatant Enzyme in Triton-Treatment Enzyme extract of ppt

A per 10 min per 20 mg fresh wt

Zero time 0.580 0.08624 hr H20 0.580 (100)1 0.102 (100)24 hr ABA 0.640 (110) 0.302 (350)24 hr ABA + chromomycin 0.620 (100) 0.170 (200)24 hr ABA + cycloheximide 0.580 (100) 0.040 (47)24 hr ABA + 6-methylpurine 0.610 (100) 0.094 (100)

1 Figures in parentheses express enzyme activity in percent ofzero time.

in acid phosphatase caused by ABA in 6 hr was inhibited com-pletely by cycloheximide or 6-methylpurine.

Substrate Specificity of Acid Phosphatase. For the acid phos-phatase that is extracted from the precipitate fraction with TritonX-100 the relative affinities for some natural phosphomonoestersas compared with that for p-NPP is as follows: ATP, UTP, andp-NPP (1.1) > ADP (0.8) > glycerophosphate (0.3) > glucose-6-phosphate (0.13) > phosphorylethanolamine (0.08). The rateof cleavage of these substrates by the supernatant enzyme wassimilar quantitatively and qualitatively to that of the Triton-ex-tractable enzyme. The relative affinity shown by banana acidphosphatase for these substrates (4) is similar to that of the Rhoeoenzyme.

DISCUSSION

Both RNase and acid phosphatase activity increase duringwater aging of Rhoeo leaf sections. The increase in RNase activityoccurs rapidly upon cutting, whereas acid phosphatase increasesabout 3-fold within a 24-hr period, beginning 24 hr after cuttingof sections. Since the activity of both RNase and acid phosphataseis inhibited by 6-methylpurine or chromomycin A3, and RNaseto a lesser extent by 5-FU, it may be concluded that increased en-

zyme activity is dependent upon synthesis of some form of RNAor specific messenger RNAs for both RNase and acid phos-phatase. The increase in activity of both enzymes is inhibited alsoby cycloheximide. This could indicate that the increased activityis owed to de novo synthesis of these enzymes, or of an enzyme pro-tein needed for activation of a precursor of one or both of theseenzymes. Another possibility is that increased activity of RNaseand acid phosphatase is owed to an accumulation of these en-zymes as a result of a specific decrease in the rate of their degrada-tion (7).Some points may be noted in consideration of an evaluation of

the significance of the hormonal regulation of activity of RNaseand acid phosphatase in relation to hormonal regulation ofsenescence. The increases in RNase and acid phosphatase are notpart of a general increase in protein synthesis during aging of leafsections, as it has been shown (14) that within 24 to 48 hr ofaging there occurs a decline in total protein. Thus, the effects ofauxin and ABA on RNase and acid phosphatase appear likehormonal effects on specific enzymes. For example, treating tissuesections with ABA increases activity of RNase at the same timethat it inhibits incorporation of uridine and leucine, whereasauxin treatment suppresses RNase activity and the effects of ABA,while enhancing incorporation of these precursors. Work withextracted enzyme indicates that these effects are not explicable interms of ABA and auxin acting as positive and negative allostericeffectors of enzyme activity respectively, nor is auxin suppressionof RNase owed to auxin mediating synthesis of an inhibitor of theenzyme. Cycloheximide and 6-methylpurine inhibit the ABA-in-duced increase in RNase and acid phosphatase. Thus, it is possiblethat ABA and auxin affect the synthesis of RNase and acid phos-phatase, perhaps at the level of transcription. Regardless of themode of hormone action, the fact that the level of acid phospha-tase and RNase in leaf sections is affected by auxin and ABA sug-gests the possibility that these hormones have a similar regulatoryrole during senescence of intact leaves. Auxin suppression of theeffects of ABA illustrates an opposing action among auxin andABA in regulation of enzyme level, as is the case for gibberellinand ABA in barley aleurone (3) and kinetin and ABA during leafsenescence (18), (Elsa Hodge and J. A. Sacher, unpublished re-sults). An interesting point of difference is that in barley aleuroneABA inhibits synthesis of hydrolases (amylase, RNase, and pro-tease) while it enhances the level of hydrolases (RNase and acidphosphatase) in leaf sections.The effects of auxin in suppressing and ABA in enhancing ac-

tivity of acid phosphatase, increases in free space and the ultimatesenescence of Rhoeo leaf sections are abiding and independent ofthe growth conditions preceding removal of leaves. Also, the in-creased level of acid phosphatase does not occur until 24 hr aftercutting of leaf sections. Thus, the development of acid phos-phatase seems like a natural senescent change, i.e., not a result ofwounding. Consistent with this view is a similar association of alarge increase in the amount of a soluble and a precipitate-boundacid phosphatase in banana pulp (4), banana peel, and avocado (J.A. Sacher, unpublished results) attending the onset of the climac-teric in both whole fruit and slices. The possible consequences ofan increase in acid phosphatase for senescence have been dis-cussed (4).

Acknowledgments-We express appreciation to Mrs. Elizabeth Anderson and Mr.George Wong for valuable technical assistance and thank Union Carbide Corp. forsamples of Carbowax. We are particularly indebted to Dr. 0. E. Smith for providingus with a supply of abscisic acid.

LFTERATURE CITED

1. ABELES, F. B., R. E. HoLM, AND H. E. GAHAGAN. 1968. Abscission: induction ofdegradative enzymes during aging. In: F. Wightman and G. Setterfield, eds.,Biochemistry and Physiology of Plant Growth Substances, 6th InternationalConference on Plant Growth Substances. The Runge Press, Ottawa. pp. 1515-1523.

810 DE LEO AND SACHER

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HORMONAL CONTROL OF RNAsE AND ACID PHOSPHATASE

2. BAGI, G. AND G. L. FARKAS. 1967. On the nature of increase in ribonuclease ac-tivity in mechanically damaged tobacco leaf tissues. Phytochemistry 6: 161-169.

3. CHRISPEELS, M. J. AND J. E. VARNER. 1967. Hormonal control of enzyme synthesis;on the mode of action of gibberellic acid and abscisin in the aleurone layers ofbarley. Plant Physiol. 42: 1008-1016.

4. DE LEO, PIETRO and J. A. SACHEL 1970. Senescence. Association of synthesis ofacid phosphatase with banana ripening. Plant Physiol. 46: 208-211.

5. DovE, L. D. 1967. Ribonuclease activity of stressed tomato leaflets. Plant Physiol.42: 1176-1178.

6. EL-ANTABLY, H. M. M., P. F. WAREING, AND J. HILLMAN. 1967. Some physiolog-ical responses to D, L-abscisin (dormin). Planta 73: 74-90.

7. FILNER, P, J. L. WRAY, AND J. E. VARNER. 1969. Enzyme induction in higherplants. Science 165: 358-367.

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Plant iThysiol. Vol. 46, 1970

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